Nature Communications
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2025
·Vol. 16
·期 1
·1-16
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IF 15.7
Fang Lixia,Hao Xueyan,Fan Jie,Liu Xiaolei,Chen Yaru,Wang Lian,Huang Xiaoying,Song Hao,Cao Yingxiu
Microbial physiology plays a pivotal role in construction of superior microbial cell factories for efficient biosynthesis of desired products. Here we identify that pcnB repression confers improved physiology for overproduction of free fatty acids (FFAs) in Escherichia coli through genome-scale CRISPRi modulation combining fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS). The repression of pcnB can enhance the stability and abundance of the transcripts of genes involved in the proton-consuming system, thereby supporting global improvements in membrane properties, redox state, and energy level. Based on pcnB repression, further repression of acrD increases FFAs biosynthesis by enhancing FFAs efflux. The engineered strain pcnB i - acrD i - fadR + achieves 35.1 g L −1 FFAs production in fed-batch fermentation, which is the maximum titer reported to date in E. coli . This study highlights the significance of uncovering hidden genetic determinants that confer improved microbial physiology for enhancing the biosynthesis of desired products.
研究领域:
合成生物学
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微生物学
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代谢工程
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PubMed
高影响因子
JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH
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2024
·Vol. 43
·期 1
·1-16
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IF 11.4
Li Xing,Sun Shiyu,Zhang Wansong,Liang Ziwei,Fang Yitong,Sun Tianhu,Wan Yong,Ma Xingcong,Zhang Shuqun,Xu Yang,Tian Ruilin
Background Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. Current treatment options are limited and often ineffective. CAR T cell therapy has shown success in treating hematologic malignancies, and there is growing interest in its potential application in solid tumors, including GBM. However, current CAR T therapy lacks clinical efficacy against GBM due to tumor-related resistance mechanisms and CAR T cell deficiencies. Therefore, there is a need to improve CAR T cell therapy efficacy in GBM. Methods We conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 MG cells co-cultured with B7-H3 targeting CAR T cells to identify genetic modifiers that can enhance CAR T cell-mediated tumor killing. Flow cytometry-based tumor killing assay and CAR T cell activation assay were performed to validate screening hits. Bioinformatic analyses on bulk and single-cell RNA sequencing data and the TCGA database were employed to elucidate the mechanism underlying enhanced CAR T efficacy upon knocking down the selected screening hits in U87 MG cells. Results We established B7-H3 as a targetable antigen for CAR T therapy in GBM. Through large-scale CRISPRi screening, we discovered genetic modifiers in GBM cells, including ARPC4 , PI4KA , ATP6V1A , UBA1 , and NDUFV1 , that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, we discovered that TNFSF15 was upregulated in both ARPC4 and NDUFV1 knockdown GBM cells and revealed an immunostimulatory role of TNFSF15 in modulating tumor-CAR T interaction to enhance CAR T cell efficacy. Conclusions Our study highlights the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifies potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Furthermore, we devised targeted strategies that synergize with CAR T therapy against GBM. These findings shed light on the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors.
CAS / MDL:
研究领域:
肿瘤学
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免疫学
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基因组学
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细胞生物学
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PubMed
高影响因子
Journal of Translational Medicine
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2025
·Vol. 23
·期 1
·1-18
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IF 7.5
Li Xing,Zhang Wansong,Fang Yitong,Sun Tianhu,Chen Jian,Tian Ruilin
Background Glioblastoma (GBM) patients frequently develop resistance to temozolomide (TMZ), the standard chemotherapy. While targeting cancer metabolism shows promise, the relationship between metabolic perturbation and drug resistance remains poorly understood.Methods We performed high-throughput CRISPR interference screens in GBM cells to identify genes modulating TMZ sensitivity. Findings were validated using multiple GBM cell lines, patient-derived glioma stem cells, and clinical data. Molecular mechanisms were investigated through transcriptome analysis, metabolic profiling, and functional assays.Results We identified phosphoglycerate kinase 1 (PGK1) as a key determinant of TMZ sensitivity. Paradoxically, while PGK1 inhibition suppressed tumor growth, it enhanced TMZ resistance by inducing metabolic stress. This activated AMPK and HIF-1α pathways, leading to enhanced DNA damage repair through 53BP1. PGK1 expression levels correlated with TMZ sensitivity across multiple GBM models and patient samples.Conclusion sOur study reveals an unexpected link between metabolic stress and chemoresistance, demonstrating how metabolic adaptation can promote therapeutic resistance. These findings caution against single-agent metabolic targeting and suggest PGK1 as a potential biomarker for TMZ response in GBM.
研究领域:
癌症生物学
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分子生物学
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基因组学
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转化医学
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DOI
PubMed
高影响因子
BIORESOURCE TECHNOLOGY
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2025
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IF 9
Yang Li,Changyang Yang,Mingxiong Liu,Hongxin Fu,Jufang Wang
Biomanufacturing offers a promising alternative for the production of the building block of nylons, of which butyrolactam is an important platform chemical. To date, there is few studies on butyrolactam biosynthesis, and static control characterized by orientation, irreversibility and ineffectiveness often impedes synthesis efficiency. Here, the chassis Escherichia coli was systematically engineered for butyrolactam synthesis by stepwise optimization strategies. In particular, inadequate α-ketoglutarate pool was identified as the main bottleneck, and then the CRISPRa and CRISPRi served as ON- and OFF-switch for establishing the temperature-powered bioswitch (thereafter renamed AITB circuit), achieving simultaneous transcriptional down- and up-regulation of the multi-genes. In the fed-batch culture, the highest titer of 59.87 g/L butyrolactam was produced by redirecting α-ketoglutarate catabolism. Further, with cassava starch as the substrate, a total of 14.96 g/L butyrolactam was generated, providing the possibility for bioproduct synthesis from renewable feedstock. This study provides efficient tool for developing high-performance biorefineries for bio-based compounds production of interest.
研究领域:
合成生物学
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代谢工程
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生物技术
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DOI
PubMed
高影响因子
TRENDS IN BIOTECHNOLOGY
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2025
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IF 14.9
Panpan Wu,Zhenyue Xu,Zhongqiu Meng,Ketao Chen,Hao Lin,Min Zhang,Lixin Zhang,Buchang Zhang,Hang Wu
Nicotinamide adenine dinucleotide (NAD) homeostasis is crucial for secondary metabolism in antibiotic-producing actinomycetes. However, NAD-based dynamic control strategies for boosting antibiotic titers have not been reported. We identified SACE_1905, an alcohol dehydrogenase in Saccharopolyspora erythraea , which converts NADH to NAD + . Overexpressing SACE_1905 lowered the cellular NADH/NAD + ratio, facilitating carbon source utilization and erythromycin biosynthetic precursor supply, concurrently improving cell growth and erythromycin yield. To balance primary and secondary metabolism, we developed a strategy that fine-tunes SACE_1905 expression with inducible clustered regularly interspaced short palindromic repeat interference (CRISPRi) to dynamically modulate the NADH/NAD + ratio, which we named diNAD. Optimized diNAD redirected carbon flux, maximizing erythromycin biosynthesis at a moderate NADH/NAD + ratio during the stationary phase. Based on its utility in actinorhodin and avermectin overproduction in Streptomyces coelicolor and Streptomyces avermitilis , we show that diNAD is effective in augmenting antibiotic titers in actinomycetes.
研究领域:
微生物学
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生物技术
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合成生物学
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生物工程
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分子生物学
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DOI
PubMed
高影响因子
Nature Communications
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2025
·Vol. 16
·期 1
·1-19
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IF 15.7
Zhang Yu,Zhang Tingting,Xiao Xue,Wang Yejun,Kawalek Adam,Ou Jinzhao,Ren Anmin,Sun Wenhao,de Bakker Vincent,Liu Yujie,Li Yuelong,Yang Liang,Ye Liang,Jia Ning,Veening Jan-Willem,Liu Xue
With the rise of antibiotic-resistant bacteria, non-antibiotic therapies like gallium gain increasing attention. Intravenous gallium nitrate is under Phase II clinical trials to treat chronic Pseudomonas aeruginosa infections in cystic fibrosis patients. However, its clinical efficacy is constrained by the achievable peak concentration in human tissue. To address this limitation, we apply a genome-wide CRISPR interference approach (CRISPRi-seq) to identify potential synergistic targets with gallium. We classify the essential genes by response time and growth reduction, pinpointing the most vulnerable therapeutic targets in this species. In addition, we identify a highly conserved gene, fprB , encoding a ferredoxin-NADP⁺ reductase, whose deletion sensitizes P. aeruginosa to gallium, lowering its MIC by 32-fold and shifting mode of action from bacteriostatic to bactericidal. Further investigation reveals that FprB plays a critical role in modulating oxidative stress induced by gallium, via control of iron homeostasis and reactive oxygen species accumulation. Deleting fprB enhances gallium’s efficacy against biofilm formation and improves outcomes in a murine lung infection model of P. aeruginosa , suggesting FprB is a promising drug target in combination with gallium. Overall, our data show CRISPRi-seq as a powerful tool for systematic genetic analysis of P. aeruginosa , advancing the identification of novel therapeutic targets.
研究领域:
微生物学
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分子生物学
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药理学
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基因组学
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DOI
PubMed
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
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2020
·Vol. 104
·期 18
·7787-7799
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IF 3.53
Yu Shengzhu,Guo Liwei,Zhao Luyao,Chen Zhenya,Huo Yixin
Phloroglucinol is a three-hydroxyl phenolic compound and has diverse physiological and pharmacological activities such as antivirus and anti-inflammatory activities. Chemical synthesis of phloroglucinol suffered from many drawbacks such as high cost and environmental pollution. To avoid the above issues, microbial phloroglucinol biosynthesis was successfully accomplished in this study, while the abundant and low-cost acetate was used as the main carbon source. Firstly, the toxicity of phloroglucinol was tested, and E. coli BL21(DE3) could tolerate 5 g/L phloroglucinol. The ability of phloroglucinol synthase (PhlD) for catalyzing malonyl-CoA to phloroglucinol was confirmed, and E. coli BL21(DE3) expressing PhlD and acetyl-CoA carboxylase (ACCase) could produce 1107 ± 12 mg/L phloroglucinol from glucose. Then, E. coli BL21(DE3) was engineered to utilize acetate to produce 228 ± 15 mg/L phloroglucinol. Then, the endogenous citrate synthase (GltA) which could catalyze oxaloacetate and acetyl-CoA generated from acetate to citrate was knocked down by CRISPRi system in order to enhance the carbon flux for phloroglucinol production, and the titer was improved to 284 ± 8 mg/L. This work demonstrated that acetate could be used as low-cost substrate to achieve the biosynthesis of phloroglucinol and provided an example of effective utilization of acetate.
研究领域:
生物技术
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代谢工程
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微生物生物技术
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DOI
PubMed
高影响因子
Synthetic and Systems Biotechnology
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2022
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IF 4.692
Ming Zhao,Yuting Li,Fengqing Wang,Yuhong Ren,Dongzhi Wei
Itaconic acid (ITA), an effective alternative fossil fuel, derives from the bypass pathway of the tricarboxylic acid (TCA) cycle. Therefore, the imbalance of metabolic flux between TCA cycle and ITA biosynthetic pathway seriously limits the production of ITA. The optimization of flux distribution between biomass and production has the potential to the productivity of ITA. Based on the previously constructed strain Escherichia coli MG1655 Δ1-SAS-3 (ITA titer: 1.87 g/L), a CRISPRi-mediated self-inducible system (CiMS), which contained a responsive module based on the ITA biosensor Yp ItcR/P ccl and a regulative CRISPRi-mediated interferential module, was developed to regulate the flux of the TCA cycle and to enhance the capacity of the strain to produce ITA. First, a higher ITA-yielding strain, Δ4-P rmd -SAS-3 (ITA titer: 3.20 g/L), derived from Δ1-SAS-3, was constructed by replacing the promoter P J23100 , for the expression of ITA synthesis genes, with P rmd and knocking out the three bypass genes poxB , pflB , and ldhA . Subsequently, the CiMS was used to inhibit the expression of key genes icd , pykA , and sucCD to dynamically balance the metabolic flux between TCA cycle and ITA biosynthetic pathway during the ITA production stage. The constructed strain Δ4-P rmd -SAS-3 under the dynamic regulation of the CiMS, showed a 23% increase in the ITA titer, which reached 3.93 g/L. This study indicated that CiMS was a practical strategy to dynamically and precisely regulated the metabolic flux in microbial cell factories.
研究领域:
生物技术
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代谢工程
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合成生物学
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DOI
PubMed
高影响因子
Food Bioscience
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2021
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IF 4.24
Bo Zhang,Li Chen,Jie-Yi Jin,Na Zhong,Xue Cai,Shu-Ping Zou,Hai-Yan Zhou,Zhi-Qiang Liu,Yu-Guo Zheng
: As the key precursor of coenzyme A synthesis in vivo , D-pantothenic acid plays an important role in maintaining biological function, which is widely applied in the food , pharmaceutical, and cosmetic industries. At present, it was mainly produced by chemical methods, with serious environmental pollution, therefore, it is urgent to develop an economical, practical, and environmentally friendly biological fermentation method based on microbial chassis cells. Herein, based on our previously developed D-pantothenic acid producing strains, we obtained higher yielding D-pantothenic acid producing strains by systematic metabolic engineering . First, according to the results of CRISPRi experiments, lpd were overexpressed, disrupted the non-PTS (non-phosphoenolpyruvate-pyruvate transport system) as well as enhanced the PTS (phosphoenolpyruvate-pyruvate transport system) allowing precursor accumulation. Subsequently, based on the analysis of metabolic intermediates , the pyruvate bypass pathway was blocked as well as the (R)-pantothenic acid pathway was further enhanced, increasing the titer of D-pantothenic acid to 4.60 g/L. Finally, the organic acid and branched-chain amino acid synthesis pathways were blocked, and in order to accelerate the synthesis of acetolactate and the output of D-pantothenic acid, heterologous acetolactate synthase and pantothenate transfer protein were introduced, which significantly increased the yield of D-pantothenic acid, reach to 6.33 g/L. The final strain in a 5 L bioreactor produced 32.32 g/L D-pantothenic acid with a productivity of 2.33 g/L/h and the high-yielding strain provides an effective platform for biological fermentation to produce D-pantothenic acid.
研究领域:
生物技术
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代谢工程
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微生物学
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发酵技术
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产品关联
DOI
高影响因子
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